The current 23-valent pneumococcal polysaccharide (PS) vaccine induces high antibody response in adults, but the immunogenicity of most PSs in the vaccine is low in young children. One approach for enhancing the antibody response in children is development of PS-protein conjugate vaccines for selected types. Several pneumococcal conjugate vaccines have been prepared and are currently in clinical trials. Several investigators have examined the possibility of using pneumococcal cell-surface proteins, such as pneumolysin, autolysin, pneumococcal surface protein A (PspA as carriers. Use of a cell-surface proteins as the carrier protein may provide a cross-protective antigen for prevention of pneumococcal infection. The overall objective of the present project is to examine cross-protective immunity of pneumococcal PSs conjugated with pneumococcal cell-surface protein carriers. Inactivated type 19A pneumolysin was prepared using recombinant E. coli containing the pneumolysin gene (ply). Type 19A ply was amplified by PCR reaction, inserted into pCR-blunt vector, and transformed into competent cells. The plasmid ply DNA was then inserted into pET-24b(+) vector, and transformed into competent E. coli cells. The ply gene was mutated by deletion of the nucleotides of type 19A ply gene that encode seven amino acid residues the from C-terminal. Pneumolysin was produced in cell lysate at 2-5 hrs after addition of IPTG. The cell lysate was passed through a resin affinity column and eluted with a concentration gradient of imidazole. The hemolytic activity of pneumolysin obtained from r-E. coli containing wild type ply gene is the same as that isolated from type 19A pneumococci. Hemolytic activity of mutated pneumolysin decreased more than 99%. Mutated pneumolysin having molecular weight of 56,000 was detected by the immunoblot analysis using a rabbit anti-pneumolysin serum. Pneumococcal type 9V PS- and type 19F PS-ply conjugates have been prepared by the following methods: (a) Schiff base formation and reductive amination; or (b) Conjugation with couping reagents of polyethylene glycol and polyvinylpyrrolidone. For conjugate characterization PS to protein ratios and molecular size profiles were analyzed. Groups of young mice are being immunized with 5 ug of 9V PS-ply or 19F PS-ply conjugate. We soon hope to show that cross-protection and effectiveness of pneumococcal conjugate vaccines can be improved by supplementation with pneumococcal cell-surface proteins as carrier for PS conjugates.